Methods and compositions for economically synthesizing and assembling long DNA sequences

Abstract

The present invention relates to a method for synthesizing and assembling long DNA sequences from short synthetic oligonucleotides. More specifically, the present invention is a cost-effective method for producing large segments of DNA of interest.

We claim:  
     
         1 . A method for producing a biologically functional DNA sequence of greater than 200 bases long comprising the steps of: 
 (a) synthesizing on a substrate an array of overlapping oligonucleotides from 10 to 200 bases encoding for either sense or antisense strand of said biologically functional DNA sequence wherein said oligonucleotides are covalently attached to the substrate using a cleavable linker;    (b) cleaving said oligonucleotides from the substrate; and    (c) assembling the mixture of overlapping oligonucleotides into said biologically functional DNA sequence.    
     
     
         2 . The method according to  claim 1  wherein said overlapping oligonucleotides are from 30 to 100 bases long.  
     
     
         3 . The method according to  claim 1  wherein the length of said biologically functional DNA sequence ranges from 200 to 10,000 bases.  
     
     
         4 . The method according to  claim 3  wherein the length of said biologically functional DNA sequence ranges from 400 to 5,000 bases.  
     
     
         5 . The method according to  claim 1  wherein said cleavable linker is a succinate like compound.  
     
     
         6 . The method according to  claim 1  wherein the number of overlapping oligonucleotides in the array is from 10 to 10,000.  
     
     
         7 . The method according to  claim 6  wherein the number of overlapping oligonucleotides in the array is from 100 to 5,000.  
     
     
         8 . The method according to  claim 1  wherein assembling the mixture of oligonucleotides further comprising enzymatic ligation.  
     
     
         9 . The method according to  claim 1  wherein assembling the mixture of oligonucleotides further comprising PCR technology.  
     
     
         10 . The method according to  claim 1  wherein assembling the mixture of oligonucleotides further comprising hybridization.  
     
     
         11 . The method according to  claim 1  wherein said biologically functional DNA sequence encodes a gene.  
     
     
         12 . The method according to  claim 1  wherein said biologically functional DNA sequence is a plasmid.  
     
     
         13 . The method according to  claim 1  wherein said biologically functional DNA sequence is a virus.  
     
     
         14 . The method according to  claim 1  wherein said biologically functional DNA sequence is the genome of an organism.  
     
     
         15 . A biologically functional DNA sequence recovered according to the method of  claim 1 .  
     
     
         16 . A substrate containing a cleavable linker for oligonucleotide synthesis according to the method of  claim 1 .  
     
     
         17 . A method for optimizing the function of a DNA sequence comprising the steps of: 
 (a) synthesizing on a substrate an array of overlapping oligonucleotides from 10-200 bases encoding for either sense or antisense strand of said DNA sequence wherein said oligonucleotides are covalently attached to the substrate using a cleavable linker;    (b) cleaving said oligonucleotides from the substrate;    (c) assembling the mixture of oligonucleotides into said DNA sequence;    (d) testing the function of said DNA sequence; and    (e) repeating the steps of (a)-(d) by varying said DNA sequence to optimize the function.